Mol Biol Rep DOI 10.1007/s11033-013-3015-3
Cloning and characterization of Na+/H+ antiporter (LfNHX1) gene from a halophyte grass Leptochloa fusca for drought and salt tolerance Muhammad Rauf • Khurram Shahzad • Rashid Ali • Moddassir Ahmad Imran Habib • Shahid Mansoor • Gerald A. Berkowitz • Nasir A. Saeed
•
Received: 27 June 2013 / Accepted: 30 December 2013 Ó Springer Science+Business Media Dordrecht 2014
Abstract Abiotic stresses such as salinity and drought have adverse effects on plants. In the present study, a Na?/ H? antiporter gene homologue (LfNHX1) has been cloned from a local halophyte grass (Leptochloa fusca). The LfNHX1 cDNA contains an open reading frame of 1,623 bp that encodes a polypeptide chain of 540 amino acid residues. LfNHX1 protein sequence showed high similarity with NHX1 homologs reported from other halophyte plants. Amino acid and nucleotide sequence similarity, protein topology modeling and the presence of conserved functional domains in the LfNHX1 protein sequence classified it as a vacuolar NHX1 homolog. The overexpression of LfNHX1 gene under CaMV35S promoter conferred salt and drought tolerance in tobacco plants. Under drought stress, transgenic plants showed higher relative water contents, photosynthetic rate, stomatal conductance and membrane stability index as compared to wild type plants. More negative value of leaf osmotic potential was also observed in transgenic plants when compared with wild type control plants. Transgenic plants showed better germination and root growth at 2 mg L-1 Basta herbicide and three levels (100, 200 and 250 mM) of sodium chloride. These results M. Rauf K. Shahzad M. Ahmad I. Habib S. Mansoor N. A. Saeed (&) Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan e-mail: [email protected] M. Rauf K. Shahzad Pakistan Institute for Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan R. Ali G. A. Berkowitz Agricultural Biotechnology Laboratories, Department of Plant Science, University of Connecticut, Mansfield, CT, USA
showed that LfNHX1 is a potential candidate gene for enhancing drought and salt tolerance in crops. Keywords Drought Leptochloa fusca Na?/H? antiporter Salt tolerance Tobacco
Introduction Plants constantly face threat from diverse biotic and abiotic factors. Among these, salinity and drought are emerging threats that affect various critical physiological, biochemical and molecular processes require for plant growth and productivity [1]. Several physiological characters have been identified and reported to contribute in plant growth under salt and osmotic stress [2, 3]. Salt stress affects the plant growth by impairing metabolism and photosynthetic activity by causing an osmotic imbalance as a result of high concentration of Na? in the cytosol. Plant can adapt under salt stress through osmotic adjustment which is characterized by cellular ion homeostasis, intercellular uptake of Na? and Cl- and vacuolar sequestration of toxic ions from cytosol [4]. Molecular mechanisms involved in abiotic stress tolerance are characterized by activation and regulation of stress related genes. Such genes are reported to be involved in signaling, transcriptional control, membrane and protein protection, ion homeostasis and scavenging of toxic ions and free radicals [5]. Vacuolar compartmentalization of toxic ions from cytoplasm facilitates functions of specific ions ratios as signal determinants and is largely achieved by H?-ATPase, located on plasma membrane or ATPase and H?-pyrophosphatase, located on tonoplast. These pumps create a H? electrochemical potential across membranes and provide driving force for secondary active transport by various transporters [6]. Two types of
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antiporters are involved in Na? efflux and compartmentalization. The first is plasma membrane Na?/H? antiporter which is also called SOS1 the second is tonoplast Na?/H? antiporter, commonly known as NHX1 [7, 8]. The NHX1 transporters are the first family of cation/proton exchangers studied in plants. The AtNHX1 was identified as an important protein involved in salt tolerance and was suggested to compartmentalize Na? in vacuoles [7]. Subsequently a large number of Na?/H? antiporter homologs have been identified and isolated from different plant species including different halophytic plants i.e. Atriplex gmelini [9], Suaeda salsa [10], Halostachys caspica [11], Salicornia brachiata [12] and Karelinia caspica [13]. Due to overexpression of NHX1, drought and salt tolerance was reported in transgenic Arabidopsis [4, 14], transgenic rice [15] and transgenic Arachis hypogaea. These results provided valid evidence that NHX1 plays a vital role in vacuolar compartmentalization of Na? and subsequently salt tolerance of plants. Leptochloa fusca is a halophyte plant from the Poaceae family and is locally known as kallar grass (grass to ameliorate salinity). It is a perennial grass which is cultivated as a fodder in many parts of Pakistan and India. Kallar grass has a lavish root system that improves the structure and permeability of soil and creates acidic conditions in the rhizosphere. L. fusca is known to tolerate high pH, sodicity, waterlogging and salinity and has both Na? accumulating and excreting properties. Due to these properties, this plant is grown for improvement of growing conditions for other salt sensitive plants [16, 17]. Up-regulation of NHX1 gene expression was also reported in L. fusca plant when grown under higher salinity levels [18] which also support this idea that Na?/H? antiporter gene may be involved in Na? accumulation properties of this plant. LfNHX1 gene isolated from this grass could be a good candidate gene for enhancing salinity and drought tolerance in various plant including major crops like wheat, cotton etc.
Materials and methods RNA isolation and cDNA synthesis Leptochloa fusca plants and salt affected soil were taken from Salinity Research Station, Pakka Ana, Toba Tek Singh, Pakistan. Plants were transplanted in pots filled with salt affected soil. Saline water was used to irrigate the plants. Total RNA was isolated from fresh leaves of L. fusca following Trizole reagent method. cDNA was synthesized using CloneMinerTM cDNA Library Construction Kit (Invitrogen, USA). cDNA library was synthesized using GatewayÒ vector pDONRTM222. Gateway PB
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reaction was performed to produce an entry library using BP ClonaseTM enzyme mix. After PB reaction, the entry library was transformed into Escherichia coli electrocompetent cells and stored at -80 °C. Polymerase chain reaction (PCR) based screening of Leptochloa fusca cDNA library PCR based cDNA library screening method was followed for isolation of the gene. Hundred microliters of library culture was spread on LB agar plates containing Kanamycin as selection marker. Due to high efficiency of cDNA library, a mat of colonies appeared on plate. All colonies were carefully scratched with the help of a scalpel and poured in 10 mL LB broth containing 50 mg mL-1 Kanamycin in Falcon tubes. Culture was stored at 4 °C for further use as template in PCR. Isolation of NHX1 gene from Leptochloa fusca cDNA library Two pairs of degenerate primers were designed from sequence alignment of NHX1 DNA sequences of different plants. One pair of primer was designed from 50 region to be used as reverse primer (50 -TGTGATYGYCATGAAA TTCCGGAAG-30 ) along with M13 forward primer (50 GTAAAACGACGGCCAG-30 ). The other primer pair was designed from 30 region to be used as forward primer (50 -C AGGTRARGAARAARCARTTCTTCCGGAATTTC-30 ) in combination with M13 reverse primer (50 -CAGGAAACAGCTATGAC-30 ). PCR profile was set as follows: 94 °C for 5 min (one cycle) followed by 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1.5 min (35 cycles) and final extension was set at 72 °C for 10 min. PCR was performed using BIORAD thermal cycler. Sequencing of PCR product was performed using DNA sequencing facility available at Bio Services Center, University of Connecticut, USA. DNA sequence was blasted using online NCBI Blast facilities. A 700 bp sequence of NHX1 starting from ATG was confirmed which showed sequence homology with already reported NHX1 sequences from various plant sources. Different pairs of degenerate primers were designed to amplify 30 end. One set of forward primer was designed to confirm sequence of LfNHX1. Forward primers were used in combination with reverse primers of 30 end. Confirmation of full-length cDNA clone of LfNHX1 For the isolation and cloning of full-length LfNHX1 gene from cDNA of L. fusca, gene specific primers (Forward: 50 -AT GGGCCCCGGCGTGGTGGC-30 ) and (Reverse 50 -TCACC GTGCTCCGTGGATGCTCTGCTCGGTGGGCG-30 ) were
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used. The amplified fragment was confirmed by gel electrophoresis and sequenced.
selection of putative transgenic plants. Putative transgenic callus and plants were selected on 2 mg L-1 Basta.
Bioinformatics analysis of LfNHX1 gene and protein
Plant transformation
LfNHX1 protein sequence was analyzed using different online tools. The sequence similarity search was performed using BLAST (Basic Local Alignment Search Tool available at NCBI website). Sequence alignment and phylogenetic analysis was done by using CLC Sequence Viewer 6.5 software. Full length LfNHX1 DNA sequence was submitted to NCBI gene bank. The PROSITE patterns PROSITE-ExPASy online bioinformatics tool was used for protein analysis. Phylogenetic analysis was performed by using software CLC Sequence Viewer 6.5. Protein secondary structure was predicted and two dimensional models of alpha helical transmembrane proteins was created by using online available software TMRPres2D [19]. Hydrophobicity plot of LfNHX1 protein sequence was predicted by online available software TMpred which is available at http://www.ch.embnet.org/software/TMPREDform.html [20]. Protein 3D structure was predicted by using online software ESyPred3D [21] available at (http:// www.fundp.ac.be/sciences/biologie/urbm/bioinfo/esypred/). The final three dimensional model was constructed by using 3D model using Chimera 1.6.2 software [22]. Protein structures was predicted on the basis of conservation scores of residues on protein structures using the ConSurf Server available at http://consurftest.tau.ac.il [23].
LfNHX1 under CaMV35S promoter was transformed in Nicotiana tabacum by Agrobacterium-medicated transformation. The cotyledon explants were co-cultivated with Agrobacterium strain AGL1 containing LfNHX1 gene under CaMV35S promoter.
Construction of entry clone For the directional cloning, CACC sequence was added to forward primer. Full length LfNHX1 was amplified using 50 -CACCATGGGCCCCGGCGTGGTGGC-30 as forward and 50 -TCACCGTGCTCCGTGGATGCTCTGCTCGGTG GGCG-30 as reverse primer. TaKaRa LA Taq proofreading DNA polymerase (Clontech, USA) was used for DNA amplification. PCR profile was set as follows: 94 °C for 5 min (one cycle), 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1.5 min (35 cycles) and final extension was set at 72 °C for 10 min. Putative PCR product was eluted and cloned into pENTRTM/D-TOPOÒ vector (Invitrogen, USA). Entry clone was confirmed by sequencing. Cloning of LfNHX1 gene in plant expression vector LfNHX1 was cloned under CaMV35S promoter in pB7WG2D,1 vector purchased from VIB (University of Gent, Belgium). Gateway LR reaction was performed to sub clone LfNHX1 from entry clone in pB7WG2D,1. The destination vector express bar gene under nos promoter for
Basta leaf paint assay For rapid screening of transgenic plants, Basta leaf bioassay was performed. Basta @ 0.05 % solution in water (w/ v) was prepared. Leaf bio-assay was performed by two different methods. In case of attached leaf method, Basta was applied on leaf surface with the help of cotton swab. In detached leaf method, leaves of transgenic and wild type plants were cut and put in Eppendorf tubes containing 0.05 % Basta solution. DNA isolation and PCR confirmation of transgenic plants DNA was isolated from the leaves of putative transgenic and wild type (WT) plants. Integrity of DNA was measured by running 2 lg DNA on 1 % agarose gel and quantity was determined with the help of spectrophotometer. PCR was performed to confirm LfNHX1 transgenic plants. Bar gene specific forward (50 -GTACCGGCAGGCTGAAGTC-30 ) and reverse (50 -GAAGTCCAGCTGCCAGAAAC-30 ) primers were used to confirm transgenic plants. Standard PCR reaction mixture was carried out. The profile used was as follows: 94 °C for 5 min (one cycle), 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1.5 min (35 cycles) and final extension was set at 72 °C for 10 min. Southern blot hybridization Southern blot hybridization was performed for checking the gene integration and copy number analysis following the protocol described by Sambrook and Russell (2001). For this purpose, 20 lg genomic DNA of putative transgenic and wild-type plants was digested with BamHI and EcoRI, resolved on 1 % agarose gel and visualized under UV light. After electrophoresis, DNA was transferred to HybondTM N? membrane (Amersham Biosciences) through capillary transfer method. The blot was hybridized with DIG labeled probe produced by using PCR DIG Probe Synthesis Kit (Roche Applied Science, Germany) by integration of DIG-11-dUTP into bar gene template through PCR. The rest of the hybridization was performed with the
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help of DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche Applied Science, USA). The rest of the hybridization process was performed with the help of DIGHigh Prime DNA Labeling and Detection Starter Kit I (Roche Applied Science).
synthesis kit (Fermentas, Cat # K1631) according to the instructions provided by the manufacturer. For RT-PCR, 1 lL of first strand cDNA was then used as template using the following gene specific primers: LfNHX1F (50 -TTGTCAAGCACCTTTCTTGG-30 ) and LfNHX1R (50 -GCAGAAGAACACGGTGAGAA-30 ).
Total RNA extraction and RT-PCR Physiological analysis Total RNA was extracted from 100 mg young leaves of transgenic and wild type tobacco plants using the Trizol Reagent (Invitrogen, Cat # 15596-026) as described earlier. To synthesize the first strand cDNA, purified total RNA was used as template. The eukaryotic mRNA has a poly-A tail that allows oligo d-T primer to bind with it and act as a primer to synthesize the first strand cDNA. This first strand cDNA was synthesized using revert Aid H- cDNA
Fig. 1 Phylogenetic analysis of Na?/H? antiporter gene. The phylogenetic tree was generated using CLC Sequence Viewer 6.5 program. The origins of the gene sequences are as follows: SeNHX1 (AY131235), Salicornia europaea; AlNHX1 (GU199336.1), Aeleuropus lagopoides; AlNHX1 (AY825361.1), Aeleuropus littoralis; LfNHX1 (JF933902), Leptochloa fusca; ZjNHX1 (EU333827.2), Zoysia japonica; PaNHX1 (AB211145.1), Phragmites australis; ScNHX1 (DQ512716.1) Suaeda corniculata; SsNHX1 (EU073422), Salsola soda; SsNHX1 (AF370358.1), Suaeda salsa; SkNHX1
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For the characterization of LfNHX1 transgenic plants, osmotic stress was applied and different physiological characters were measured. Transpiration rate, stomatal conductance and net photosynthesis rate were determined using an open system LCA-4 ADC portable infrared gas analyzer (Analytical Development Co., Hoddesdon, England).
(AB531436.1), Salsola komarovii; SbNHX1 (EU448383.1), Salicornia brachiate; AhNHX1 (HM590627.1), Arachis hypogaea; ThNHX1 (AK353509.1) Thellungiella halophile; AtNHX1 (AK226586.1), Arabidopsis thaliana; HtNHX1 (EF159151.1), Helianthus tuberosus; OsNHX1 (AB021878.1), Oryza sativa, PutNHX1 (AB628206.1), Puccinellia tenuiflora; HvNHX1, (AB089197.1), Hordeum vulgare; PeNHX1 (GU295174.1), Phyllostachys edulis; TaNHX1 (AY040246.2, T. aestivum
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Fig. 2 Alignment of Cation binding domain of Na?/H? antiporter proteins. Aeleuropus lagopoides (ACZ97405.1), Aeleuropus littoralis (AAV80466.1), Leptochloa fusca (AEN03787.1), Zoysia japonica (ABY19311.2), Phragmites australis (BAD95562.1), Sorghum bicolor (ACD64982.2), Oryza sativa (AB021878.1), Arabidopsis thaliana (NP_198067.1), Puccinellia tenuiflora (BAK23261.1), Hordeum
Osmotic potential (Ws) Fully expanded youngest leaves were excised from transgenic and wild type plants. Leaf was shifted into 1.5 mL Eppendorf tube and stored at -80 °C for 2 weeks, thawed and sap was extracted by crushing with the help of micro mortar. Sap was collected and used for determination of osmotic potential in osmometer (Vapro 5520). Value obtained in mmol kg-1 was converted into—Mpa using following formula Ws ðMpaÞ ¼ 2:5 mmol kg1 =1; 000: Relative water content (RWC) Relative water contents of leaves from transgenic and wild type plants were measured according to method described by [24]. Fresh weight of equal sized discs of young and fully expanded leaves was determined after excision. Turgid weight of all discs was also measured after soaking the leaf discs in the distilled water for 22–24 h. All discs were dried carefully on tissue paper prior to measuring turgid weight. Dry weight was also measured after completely drying the discs for 72 h at 80 °C in the incubator. RWC was calculated using the following equation:
vulgare (BAC56698.1), Phyllostachys edulis (ADB54780.1), T. aestivum (AAK76738.2), Zea mays (NP_001105221.1), Lophopyrum elongatum (AAR17789.2), Atriplex dimorphostegia (AAO48271.1), Glycine max (AAY43006.1), Iris lacteal (AAU81619.1), Gossypium hirsutum (AAM54141.2)
RWC ¼ðfresh weight dry weight/turgid weight dry weightÞ 100: Leaf membrane stability index Leaf membrane stability index (MSI %) was measured according to the method described by [25] and modified by [26]. Leaf discs of uniform size and equal weight (0.2 g) were taken in 15 mL Falcon tubes containing 10 mL distilled water. Samples were kept at 40 °C in a water bath for 30 min and electrical conductivity (C1) was measured using EC meter. Another set of same size and weight was taken in test tubes and incubated at 100 °C in water bath for 15 min. Electrical conductivity (C2) of second set was also measured. MSI % was calculated by the following formula: MSI = (1 – C1/C2) 9 100. Germination analysis of transgenic and wild type plants For testing salt and Basta herbicide stress tolerance of transgenic tobacco plants, T0 seeds of transgenic and wild type plants were germinated on MS medium supplemented with 2 mg L-1 Basta and 100, 200 and 250 mM NaCl. After 10 days, resistant and wild type seedlings were transferred to MS medium supplement with 250 mM NaCl.
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Fig. 3 a Hydrophobicity plot of LfNHX1 gene product. The hydrophobicity values were calculated by the program TMpred available online at http://www.ch.embnet.org/software/TMPRED-form.html. b Proposed topological model of LfNHX1. Coiled portion indicates the 12 transmembrane domains. Third domain contain amiloride
binding site while cation binding domains is present in 5th and 6th domain. c Homology model of LfNHX1 protein based on homology with already reported vacuolar Na?/H? antiporter protein sequence. d 3-Dimensional model of LfNHX1 protein showing the 12 transmembrane domains and loops in various colors. (Color figure online)
Statistical analysis
sequence was confirmed as NHX1 sequence by NCBI sequence blast. For amplification of full length LfNHX1 cDNA, a gene specific primer and degenerate primers from 30 end were designed by alignment of NHX1 sequences from various monocot plants. A 1,300 bp fragment was amplified and confirmed by sequencing. Full length LfNHX1 gene was amplified by using gene specific primers. Full length LfNHX1 (Open reading frame) was cloned in Gateway pENTRTM TOPOÒ vector and then in expression vector by LR reaction.
Data were statistically analyzed for significance by performing analysis of variance (ANOVA) using Statistix 8.1 software. All means were compared for significance by using least significant difference test at 0.05 probability level [27]. Results Cloning of Leptochloa fusca Na?/H? antiporter gene (LfNHX1)
Bioinformatics analysis of LfNHX1 cDNA LfNHX1 (partial sequence) gene was amplified by screening L. fusca cDNA library using PCR based library screening method using M13 forward and reverse degenerate primers. A fragment of *750 bp was amplified by PCR. Amplified fragment was sequenced and putative
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LfNHX1 cDNA (Accession number: JF933902) contains an uninterrupted open reading frame of 1,623 bp coding for a polypeptide of 540 amino acids. LfNHX1gene sequence showed 95 % similarity with Aeluropus lagopoides, 88 %
Mol Biol Rep Table 1 The PROSITE patterns for LfNHX1: positions of important sites Positions
Predicted sites/patterns
Myristyl (N-myristoylation site) 13–18
GVLSST
60–65
GLCTGV
118–123
GAVGTM
121–126
GTMISF
154–159
GAIFSA
230–235 234–239
GVFPGL GLLSAY
284–289
GIVMSH
391–396
GLMRGA
CK2_phospho_site (casein kinase II phosphorylation site) 17–20
STSD
158–161
SATD
251–254
TDRE
475–478
SDLE
530–533
SPTE
ASN_glycosylation (N-glycosylation site) 51–54
NESI
294–297
NVTE
369–372
NLTK
PKC_phospho_site (protein kinase C phosphorylation site) 70–72 251–253
TTK TDR
298–300
SSR
302–304
TTK
371–373
TKK
380–382
TWR
460–462
SPK
490–492
SLR
Leucine_zipper (leucine zipper pattern) 257–278
LMMLMAYLSYMLAELSDLSGIL
ATP_GTP_A ATP/GTP-binding site motif A (P-loop) 335–342
ASDSPGKS
with Sorghum bicolor, 87 % with Zea mays, 86 with Oryza sativa, 85 % with Hordeum vulgare and 84 % with Triticum aestivum. To investigate the molecular evolution and phylogenetic relationship among Na?/H? antiporters in plants, Na?/H? antiporter gene sequences from both dicot and monocot plants were aligned and phylogenetic tree was constructed. LfNHX1 clustered with monocot halophyte plants. LfNHX1 gene belongs to the family of plant vacuolar protein which acts as Na?/H? exchanger (Fig. 1). The sequence alignment of cation binding domains showed the high similarity of LfNHX1 cation domain with cation domains from other monocot plants while there was difference of three amino acids with cation binding domains of NHX1 gene from dicot plants (Fig. 2). The
hydrophobicity plot showed that LfNHX1 has 12 hydrophobic peak regions and output from toppred suggested that LfNHX1 contains 12 strong transmembrane domains from inside to outside and outside to inside (Fig. 3a). The protein sequence 85-LFFIYLLPPI-95 in LfNHX1 protein sequence is highly conserved. This sequence was identified as amiloride binding site in mammals which inhibits eukaryotic Na?/H? antiporter. These properties confirmed that LfNHX1 is a vacuolar Na?/H? antiporter. Analysis of LfNHX1 protein sequence also showed three potential N-glycosylation sites and nine N-myristoylations sites. Furthermore, five sites of casein kinase II phosphorylation, eight protein kinase C phosphorylation sites and a leucine zipper pattern site were also predicted in LfNHX1 protein sequence (Table 1). Two dimensional secondary structure of LfNHX1 protein sequence predicted by TMRPres2D showed 12 transmembrane domains (Fig. 3b). Amiloride binding site was predicted in 3rd domain while cation binding domains was predicted in 5th and 6th domain. A structural homology model of LfNHX1 protein was obtained based on conservation scores of residues on protein structures of already reported vacuolar NHX1 protein sequences from various plant sources using ConSurf Server (Fig. 3c). Three-dimensional structure of LfNHX1 protein sequence was built using the 3D structure 4A01 chain ‘A’ as template which shares 87.5 % identities with LfNHX1 sequence. The protein data base (PDB) file of LfNHX1 protein sequence was obtained and 3D model was drawn using Chimera 1.6.2 software [22] (Fig. 3d). The sequence alignment of LfNHX1 gene with Arabidopsis, wheat and barley showed some differences in amino acid sequence. Some differences in cation binding domains were also observed (Fig. 4). LfNHX1 protein model structure was submitted to protein model database (PMDB) with PMDBID PM0078999. Construction and confirmation of transgenic tobacco plant Transgenic tobacco plants were developed by using PBLfNHX1 construct (Fig. 5a) through Agrobacterium mediated plant transformation. With the presence of bar gene in cassette, Basta was used as plant selectable marker. Putative transgenic plants were confirmed through PCR using bar gene specific primers (Fig. 5b). Southern blot analysis of transgenic and WT plants was also performed to check the gene integration. Results of Southern blot analysis showed single copy integration of the LfNHX1 gene in PB1, PB3 and PB8 transgenic lines (Fig. 5c). Beside this, reverse transcriptase PCR (RT-PCR) was also performed to check the expression of the LfNHX1 gene in the transgenic lines. RT-PCR results proved the expression of the LfNHX1 gene in all transgenic lines while there was no expression
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Fig. 4 Protein sequence alignment of LfNHX1 with other NHX from Arabidopsis thaliana (AtNHX1, AAT95387), T. aestivum (TaNHX1, AAK76738.2), Hordeum vulgare (HvNHX1, BAC56698.1). The sequences were analyzed by CLC Sequence Viewer 6.5 software.
The identical amino acid residues are indicated by black letters and different residues are indicated by white letters with black background. Small box shows amiloride binding site while large box shows cation binding domain
in wild type plant (Fig. 5d; top panel). By contrast, the house-keeping gene (NtActin) showed expression in all samples including wild type control (Fig. 5d; bottom panel). Confirmation of transgenic plant was also performed by two types of Basta leaf assays. These results showed prominent differences in transgenic and wild type plants. In case of attached Basta paint leaf assay, the circled areas where Basta herbicide was painted showed necrosis in wild type plants while transgenic plants remained unaffected (Fig. 6a). In case of detached leaf assay, leaves of transgenic plants remained green after 7 days while leaves of WT plant lost their chlorophyll (Fig. 6b).
potential as compare to wild type (Fig. 7d). It was observed that drought and heat stress decreased the membrane stability index (%) in wild type plants. Significant differences between transgenic lines and wild type plants were observed (P \ 0.05) which indicated that transgenic line possess higher membrane stability in case of drought cum heat stress (Fig. 7e).
Physiological analysis of plants for drought tolerance Transgenic plants were subjected to drought and heat stress. Plants were exposed to sunlight in net house during the summer season. Excessive heat caused rapid water loss and leaf desiccation. Holding irrigation for 7 days followed by re-watering caused great damage to the wild type plants while transgenic plants remained green and fresh even at 40 °C (Fig. 6c). Significant differences (P \ 0.05) were also observed for relative water contents, photosynthetic rate and stomatal conductance. Transgenic lines showed higher relative water contents, net photosynthetic rate and stomatal conductance as compared to wild type plants (Fig. 7a, b, c). In case of osmotic potential, transgenic plants showed significantly less negative value of osmotic
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Analysis of transgenic plants for salt and Basta herbicide tolerance Seeds of PB3 transgenic line and wild type plants were germinated on MS medium supplemented 2 mg L-1 Basta. Most of the transgenic seedlings were able to maintain their growth (Fig. 8a) while wild type seeds were unable to maintain growth and died after few days of germination (Fig. 8b). Transgenic plantlets also showed better root length at, 200 and 250 mM NaCl (Fig. 8c) while wild type plant were unable to maintain their normal growth and root length (Fig. 8d). Transgenic plant seed also showed segregation pattern showed restricted growth and behaved like wild type seed which were unable to maintain their normal growth after germination when germinated on MS0 supplemented with 2 mg L-1 Basta and 200 mM NaCl (Fig. 8d). Transgenic seedlings and wild type plant seedlings were also shifted to jars containing MS0 with 250 mM NaCl. Transgenic seedlings were able to maintain their normal shoot growth (Fig. 8e) and root length (Fig. 8g–i) while wild type seedlings showed leaf
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Fig. 5 a Diagrammatic representation of the LfNHX1 construct used for tobacco transformation. b PCR confirmation of T0 transgenic tobacco plants using bar primers. Lanes 1–6 were transgenic plants, lane 7 was wild type control and lane 8 was plasmid positive control. A 600 bp fragment of bar gene was amplified T0 transgenic lines while in wild type no amplification was detected. c Southern blot analysis of T0 transgenic plants. Lanes 1–5 were transgenic lines, lane 6 was wild-type and lane 7 was positive control. A 600 bp fragment of bar gene was used as a probe. d Reverse transcriptase PCR analysis
T0 transgenic plants showed expression of LfNHX1 gene. Gene specific primers were used to amplify 200 bp coding region of LfNHX1. NtActin gene was used as an internal control. Upper panel shows expression of LfNHX1 in transgenic tobacco. Lanes 1–6 shows 200 bps amplified PCR fragment in LfNHX1-PB transgenic lines while lane 7 represents wild type plant without amplification. Lower panel represents expression NtActin gene as internal control in transgenic as well as wild type plant (lanes 1–7) gene
chlorophyll loss and dryness (Fig. 8f) and restricted root growth (Fig. 8j) which indicated that LfNHX1 gene is working and involved in Na? extrusion mechanism.
generating proton pumps [29]. The basic salt tolerance mechanism is same in most plant species. Various Na?/H? antiporters homologs isolated from various plant species appeared to be functional for abiotic stress tolerance but cloning of Na?/H? antiporter gene homologs from halophytes would be particularly interesting because they might be functionally more efficient than those from glycophytes. Therefore research should be focused on isolation of Na?/ H? antiporters from various halophytic plant species which could survive under high saline conditions. Considering the importance of vacuolar Na?/H? antiporters in salt tolerance, it is assumed that the vacuolar Na?/H? antiporters from halophytes might have been active than those from glycophytes [30].
Discussion Drought and salinity adversely affect plant growth and limit crop yield. Plants can be classified into two groups based on salt tolerance. Glycophytes are salt sensitive while halophytes are salt tolerant plants [28, 29]. In comparison with glycophytes, halophytes have well developed mechanism for salt tolerance which may be due to the high efficiency of Na?/H? antiporter genes and electro gradient
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Fig. 6 a Leaf paint assay using 0.05 % Basta. b Detached leaf dip assay using 0.05 % Basta. c Drought stress was applied to transgenic PB3 and PB6 and wild type (WT) plants by stopping water for 7 days followed by rewatering on wilting
In this work, NHX1 homolog was isolated from a halophyte grass L. fusca by using different sets of degenerate primers. The amplified fragments were sequenced and confirmed as NHX1 gene by using NCBI blast. Bioinformatics analysis showed amino acid sequence similarity of LfNHX1 with reported vacuolar Na?/H? antiporters from various plants. LfNHX1 protein sequence showed twelve putative transmembrane domains like other plant NHX1 sequences such as SsNHX1 [31], GhNHX1 [32], ThNHX1 [33] and ZxNHX1 [34]. In the previous studies it was reported that the third transmembrane region of LfNHX1 contains the amiloride binding site (LFFIYLLPPI) which is conserved in eukaryotic Na?/H? antiporters and inhibit their activity in the presence of amiloride [31, 35–40]. This putative conserved amiloride binding domain (85LFFIYLLPPI-94) is also present in the TM3 at N-terminal of LfNHX1 protein sequence. The amino acid differences and cation binding domain indicate the differences of LfNHX1 protein sequence from AtNHX1 to TaNHX1 protein sequences. LfNHX1 gene was cloned under CaMV35S promoter using Gateway technology and tobacco transformation was done using Agrobacterium-mediated plant transformation method. Putative LfNHX1-transgenic plants were confirmed by PCR and copy number was observed using Southern hybridization whereas RT-PCR was performed for gene expression. Transgenic plants were also confirmed using Basta leaf assay. Visarada et al. [41] reported that transgenic plants can be easily identified by using leaf paint assay. Vengadesan et al. [42] reported that expression of the bar gene in transgenic plant can be confirmed by using a leaf painting test with herbicide Basta. Soliman et al. [43] also applied Basta leaf paint assay to select transgenic AtNHX1 plants containing bar gene as plant selectable marker. Results presented in this
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study are consistent with the previous reports. Some of the transgenic lines and control plant were subjected to osmotic stress by withholding water supply for 7 days during hot summer weather in the month of May when average daily day time temperature remains near 40 °C. Wild type (control) plants showed rapid leaf desiccation and growth inhibition than the transgenic plants. Brini et al. [4] and Wei et al. [44] also reported better leaf area growth and enhanced drought tolerance in case of transgenic Arabidopsis plants over-expressing TNHX1 gene from T. aestivum and AmNHX2 gene from Ammopiptanthus mongolicus, thus indicating the usability of LfNHX1under water deficit conditions. Transgenic lines showed more net photosynthetic rate and relative water contents, stomatal conductance more negative value of leaf osmotic potential and higher cell membrane stability index as compared to control plants. He et al. [45] reported higher photosynthetic rate in the transgenic cotton plants overexpressing AtNHX1 gene as compared to WT plant. Banjara et al. [46] showed enhanced photosynthetic rate and stomatal conductance in transgenic peanut overexpressing AtNHX1 gene under salt stress. Enhanced photosynthetic rate and stomatal conductance was also reported in popular plant overexpressing AtNHX1 gene under normal as well as low and high salt concentration [47]. Similarly more negative leaf water potential was observed in Arabidopsis thaliana plants overexpressing wheat Na?/H? antiporter TNHX1 gene that is in line with the findings of present study and may be due to the superior osmotic adjustment of LfNHX1 transgenic plants [4]. Transgenic plants showed higher seed germination than wild type under Basta and salt stress. Higher root and shoot length was observed in transgenic seedlings as compared to wild type. The LfNHX1-transgenic tobacco plants exhibited a better growth on 2 mg L-1 Basta while
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Fig. 7 Physiological analysis of transgenic and wild-type plants for drought tolerance. The response was measured in terms of: a relative water content RWC, b photosynthetic rate, c stomatal conductance,
d osmotic potential and e cell membrane stability index. Bars represent mean ± standard deviation
wild type plants did not maintain their survival after germination. Longer root and shoot growth was observed in transgenic plants when germinated on 100, 200 and 250 mM NaCl. This improved cellular expansion under salt stress may be due to the mediation of vacuolar K? and pH. Enhanced K?/H? exchange was also reported in case of knock out mutants of NHX1 and NHX2 genes in A. thaliana [48]. Overexpression of vacuolar AtNHX1 conferred tolerance to both salt and drought stresses in transgenic petunia plants [49]. In present study, vacuolar Na?/ H? antiporter from a halophyte grass L. fusca enhanced salt and drought tolerance in the LfNHX1-transgenic tobacco
plant. Our results suggested that LfNHX1 would be the candidate gene for engineering salt tolerance in crop plants. Many previous reports have established that H?-pyrophosphatase and Na?/H? antiporter work by complimenting each other during abiotic stress. Vacuolar proton generating pumps provide H? ions which are used by vacuolar Na?/H? antiporter for extrusion of Na? from cytoplasm into vacuole. There is need to focus on identification of H?-pyrophosphatase and Na?/H? antiporter gene homologs from halophyte plant species. Transcription factors and promoters from halophyte plants would be the best candidates to study the salt tolerance mechanisms.
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Mol Biol Rep Fig. 8 Germination test of LfNHX1 PB3 (T1) transgenic seeds (L) and wild type (R) plant seeds. a Germination of wild type (control) seeds. b Growth of transgenic seeds on MS0 supplement 2 mg L-1 Basta. c Germination of LfNHX1-PB3 transgenic seeds on MS0 with 200 mM NaCl for salt tolerance. d Germination of WT seeds on 250 mM NaCl. e Growth of transgenic seedlings on MS0 supplemented with 250 mM NaCl. f Growth of WT seedlings on 250 mM NaCl. g–i Root and shoot comparison of LfNHX1-PB3 transgenic seedlings on MS0 with 250 mM NaCl. j Growth of WT seedlings on 250 mM NaCl
Overexpressing H?-pyrophosphatase and Na?/H? antiporter gene homologs from halophyte under indigenous promoters would be helpful in generating abiotic stress tolerance in major staple food crops like wheat, maize etc. Acknowledgments The authors are also very thankful to Higher Education Commission (HEC) of Pakistan for providing PhD scholarship to Mr. Muhammad Rauf for conducting his PhD studies at NIBGE and his six months fellowship at Agricultural Biotechnology Laboratories University of Connecticut, USA.
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Cloning and characterization of Na+/H+ antiporter (LfNHX1) gene from a halophyte grass Leptochloa fusca for drought and salt tolerance Muhammad Rauf • Khurram Shahzad • Rashid Ali • Moddassir Ahmad Imran Habib • Shahid Mansoor • Gerald A. Berkowitz • Nasir A. Saeed
•
Received: 27 June 2013 / Accepted: 30 December 2013 Ó Springer Science+Business Media Dordrecht 2014
Abstract Abiotic stresses such as salinity and drought have adverse effects on plants. In the present study, a Na?/ H? antiporter gene homologue (LfNHX1) has been cloned from a local halophyte grass (Leptochloa fusca). The LfNHX1 cDNA contains an open reading frame of 1,623 bp that encodes a polypeptide chain of 540 amino acid residues. LfNHX1 protein sequence showed high similarity with NHX1 homologs reported from other halophyte plants. Amino acid and nucleotide sequence similarity, protein topology modeling and the presence of conserved functional domains in the LfNHX1 protein sequence classified it as a vacuolar NHX1 homolog. The overexpression of LfNHX1 gene under CaMV35S promoter conferred salt and drought tolerance in tobacco plants. Under drought stress, transgenic plants showed higher relative water contents, photosynthetic rate, stomatal conductance and membrane stability index as compared to wild type plants. More negative value of leaf osmotic potential was also observed in transgenic plants when compared with wild type control plants. Transgenic plants showed better germination and root growth at 2 mg L-1 Basta herbicide and three levels (100, 200 and 250 mM) of sodium chloride. These results M. Rauf K. Shahzad M. Ahmad I. Habib S. Mansoor N. A. Saeed (&) Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan e-mail: [email protected] M. Rauf K. Shahzad Pakistan Institute for Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan R. Ali G. A. Berkowitz Agricultural Biotechnology Laboratories, Department of Plant Science, University of Connecticut, Mansfield, CT, USA
showed that LfNHX1 is a potential candidate gene for enhancing drought and salt tolerance in crops. Keywords Drought Leptochloa fusca Na?/H? antiporter Salt tolerance Tobacco
Introduction Plants constantly face threat from diverse biotic and abiotic factors. Among these, salinity and drought are emerging threats that affect various critical physiological, biochemical and molecular processes require for plant growth and productivity [1]. Several physiological characters have been identified and reported to contribute in plant growth under salt and osmotic stress [2, 3]. Salt stress affects the plant growth by impairing metabolism and photosynthetic activity by causing an osmotic imbalance as a result of high concentration of Na? in the cytosol. Plant can adapt under salt stress through osmotic adjustment which is characterized by cellular ion homeostasis, intercellular uptake of Na? and Cl- and vacuolar sequestration of toxic ions from cytosol [4]. Molecular mechanisms involved in abiotic stress tolerance are characterized by activation and regulation of stress related genes. Such genes are reported to be involved in signaling, transcriptional control, membrane and protein protection, ion homeostasis and scavenging of toxic ions and free radicals [5]. Vacuolar compartmentalization of toxic ions from cytoplasm facilitates functions of specific ions ratios as signal determinants and is largely achieved by H?-ATPase, located on plasma membrane or ATPase and H?-pyrophosphatase, located on tonoplast. These pumps create a H? electrochemical potential across membranes and provide driving force for secondary active transport by various transporters [6]. Two types of
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antiporters are involved in Na? efflux and compartmentalization. The first is plasma membrane Na?/H? antiporter which is also called SOS1 the second is tonoplast Na?/H? antiporter, commonly known as NHX1 [7, 8]. The NHX1 transporters are the first family of cation/proton exchangers studied in plants. The AtNHX1 was identified as an important protein involved in salt tolerance and was suggested to compartmentalize Na? in vacuoles [7]. Subsequently a large number of Na?/H? antiporter homologs have been identified and isolated from different plant species including different halophytic plants i.e. Atriplex gmelini [9], Suaeda salsa [10], Halostachys caspica [11], Salicornia brachiata [12] and Karelinia caspica [13]. Due to overexpression of NHX1, drought and salt tolerance was reported in transgenic Arabidopsis [4, 14], transgenic rice [15] and transgenic Arachis hypogaea. These results provided valid evidence that NHX1 plays a vital role in vacuolar compartmentalization of Na? and subsequently salt tolerance of plants. Leptochloa fusca is a halophyte plant from the Poaceae family and is locally known as kallar grass (grass to ameliorate salinity). It is a perennial grass which is cultivated as a fodder in many parts of Pakistan and India. Kallar grass has a lavish root system that improves the structure and permeability of soil and creates acidic conditions in the rhizosphere. L. fusca is known to tolerate high pH, sodicity, waterlogging and salinity and has both Na? accumulating and excreting properties. Due to these properties, this plant is grown for improvement of growing conditions for other salt sensitive plants [16, 17]. Up-regulation of NHX1 gene expression was also reported in L. fusca plant when grown under higher salinity levels [18] which also support this idea that Na?/H? antiporter gene may be involved in Na? accumulation properties of this plant. LfNHX1 gene isolated from this grass could be a good candidate gene for enhancing salinity and drought tolerance in various plant including major crops like wheat, cotton etc.
Materials and methods RNA isolation and cDNA synthesis Leptochloa fusca plants and salt affected soil were taken from Salinity Research Station, Pakka Ana, Toba Tek Singh, Pakistan. Plants were transplanted in pots filled with salt affected soil. Saline water was used to irrigate the plants. Total RNA was isolated from fresh leaves of L. fusca following Trizole reagent method. cDNA was synthesized using CloneMinerTM cDNA Library Construction Kit (Invitrogen, USA). cDNA library was synthesized using GatewayÒ vector pDONRTM222. Gateway PB
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reaction was performed to produce an entry library using BP ClonaseTM enzyme mix. After PB reaction, the entry library was transformed into Escherichia coli electrocompetent cells and stored at -80 °C. Polymerase chain reaction (PCR) based screening of Leptochloa fusca cDNA library PCR based cDNA library screening method was followed for isolation of the gene. Hundred microliters of library culture was spread on LB agar plates containing Kanamycin as selection marker. Due to high efficiency of cDNA library, a mat of colonies appeared on plate. All colonies were carefully scratched with the help of a scalpel and poured in 10 mL LB broth containing 50 mg mL-1 Kanamycin in Falcon tubes. Culture was stored at 4 °C for further use as template in PCR. Isolation of NHX1 gene from Leptochloa fusca cDNA library Two pairs of degenerate primers were designed from sequence alignment of NHX1 DNA sequences of different plants. One pair of primer was designed from 50 region to be used as reverse primer (50 -TGTGATYGYCATGAAA TTCCGGAAG-30 ) along with M13 forward primer (50 GTAAAACGACGGCCAG-30 ). The other primer pair was designed from 30 region to be used as forward primer (50 -C AGGTRARGAARAARCARTTCTTCCGGAATTTC-30 ) in combination with M13 reverse primer (50 -CAGGAAACAGCTATGAC-30 ). PCR profile was set as follows: 94 °C for 5 min (one cycle) followed by 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1.5 min (35 cycles) and final extension was set at 72 °C for 10 min. PCR was performed using BIORAD thermal cycler. Sequencing of PCR product was performed using DNA sequencing facility available at Bio Services Center, University of Connecticut, USA. DNA sequence was blasted using online NCBI Blast facilities. A 700 bp sequence of NHX1 starting from ATG was confirmed which showed sequence homology with already reported NHX1 sequences from various plant sources. Different pairs of degenerate primers were designed to amplify 30 end. One set of forward primer was designed to confirm sequence of LfNHX1. Forward primers were used in combination with reverse primers of 30 end. Confirmation of full-length cDNA clone of LfNHX1 For the isolation and cloning of full-length LfNHX1 gene from cDNA of L. fusca, gene specific primers (Forward: 50 -AT GGGCCCCGGCGTGGTGGC-30 ) and (Reverse 50 -TCACC GTGCTCCGTGGATGCTCTGCTCGGTGGGCG-30 ) were
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used. The amplified fragment was confirmed by gel electrophoresis and sequenced.
selection of putative transgenic plants. Putative transgenic callus and plants were selected on 2 mg L-1 Basta.
Bioinformatics analysis of LfNHX1 gene and protein
Plant transformation
LfNHX1 protein sequence was analyzed using different online tools. The sequence similarity search was performed using BLAST (Basic Local Alignment Search Tool available at NCBI website). Sequence alignment and phylogenetic analysis was done by using CLC Sequence Viewer 6.5 software. Full length LfNHX1 DNA sequence was submitted to NCBI gene bank. The PROSITE patterns PROSITE-ExPASy online bioinformatics tool was used for protein analysis. Phylogenetic analysis was performed by using software CLC Sequence Viewer 6.5. Protein secondary structure was predicted and two dimensional models of alpha helical transmembrane proteins was created by using online available software TMRPres2D [19]. Hydrophobicity plot of LfNHX1 protein sequence was predicted by online available software TMpred which is available at http://www.ch.embnet.org/software/TMPREDform.html [20]. Protein 3D structure was predicted by using online software ESyPred3D [21] available at (http:// www.fundp.ac.be/sciences/biologie/urbm/bioinfo/esypred/). The final three dimensional model was constructed by using 3D model using Chimera 1.6.2 software [22]. Protein structures was predicted on the basis of conservation scores of residues on protein structures using the ConSurf Server available at http://consurftest.tau.ac.il [23].
LfNHX1 under CaMV35S promoter was transformed in Nicotiana tabacum by Agrobacterium-medicated transformation. The cotyledon explants were co-cultivated with Agrobacterium strain AGL1 containing LfNHX1 gene under CaMV35S promoter.
Construction of entry clone For the directional cloning, CACC sequence was added to forward primer. Full length LfNHX1 was amplified using 50 -CACCATGGGCCCCGGCGTGGTGGC-30 as forward and 50 -TCACCGTGCTCCGTGGATGCTCTGCTCGGTG GGCG-30 as reverse primer. TaKaRa LA Taq proofreading DNA polymerase (Clontech, USA) was used for DNA amplification. PCR profile was set as follows: 94 °C for 5 min (one cycle), 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1.5 min (35 cycles) and final extension was set at 72 °C for 10 min. Putative PCR product was eluted and cloned into pENTRTM/D-TOPOÒ vector (Invitrogen, USA). Entry clone was confirmed by sequencing. Cloning of LfNHX1 gene in plant expression vector LfNHX1 was cloned under CaMV35S promoter in pB7WG2D,1 vector purchased from VIB (University of Gent, Belgium). Gateway LR reaction was performed to sub clone LfNHX1 from entry clone in pB7WG2D,1. The destination vector express bar gene under nos promoter for
Basta leaf paint assay For rapid screening of transgenic plants, Basta leaf bioassay was performed. Basta @ 0.05 % solution in water (w/ v) was prepared. Leaf bio-assay was performed by two different methods. In case of attached leaf method, Basta was applied on leaf surface with the help of cotton swab. In detached leaf method, leaves of transgenic and wild type plants were cut and put in Eppendorf tubes containing 0.05 % Basta solution. DNA isolation and PCR confirmation of transgenic plants DNA was isolated from the leaves of putative transgenic and wild type (WT) plants. Integrity of DNA was measured by running 2 lg DNA on 1 % agarose gel and quantity was determined with the help of spectrophotometer. PCR was performed to confirm LfNHX1 transgenic plants. Bar gene specific forward (50 -GTACCGGCAGGCTGAAGTC-30 ) and reverse (50 -GAAGTCCAGCTGCCAGAAAC-30 ) primers were used to confirm transgenic plants. Standard PCR reaction mixture was carried out. The profile used was as follows: 94 °C for 5 min (one cycle), 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1.5 min (35 cycles) and final extension was set at 72 °C for 10 min. Southern blot hybridization Southern blot hybridization was performed for checking the gene integration and copy number analysis following the protocol described by Sambrook and Russell (2001). For this purpose, 20 lg genomic DNA of putative transgenic and wild-type plants was digested with BamHI and EcoRI, resolved on 1 % agarose gel and visualized under UV light. After electrophoresis, DNA was transferred to HybondTM N? membrane (Amersham Biosciences) through capillary transfer method. The blot was hybridized with DIG labeled probe produced by using PCR DIG Probe Synthesis Kit (Roche Applied Science, Germany) by integration of DIG-11-dUTP into bar gene template through PCR. The rest of the hybridization was performed with the
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help of DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche Applied Science, USA). The rest of the hybridization process was performed with the help of DIGHigh Prime DNA Labeling and Detection Starter Kit I (Roche Applied Science).
synthesis kit (Fermentas, Cat # K1631) according to the instructions provided by the manufacturer. For RT-PCR, 1 lL of first strand cDNA was then used as template using the following gene specific primers: LfNHX1F (50 -TTGTCAAGCACCTTTCTTGG-30 ) and LfNHX1R (50 -GCAGAAGAACACGGTGAGAA-30 ).
Total RNA extraction and RT-PCR Physiological analysis Total RNA was extracted from 100 mg young leaves of transgenic and wild type tobacco plants using the Trizol Reagent (Invitrogen, Cat # 15596-026) as described earlier. To synthesize the first strand cDNA, purified total RNA was used as template. The eukaryotic mRNA has a poly-A tail that allows oligo d-T primer to bind with it and act as a primer to synthesize the first strand cDNA. This first strand cDNA was synthesized using revert Aid H- cDNA
Fig. 1 Phylogenetic analysis of Na?/H? antiporter gene. The phylogenetic tree was generated using CLC Sequence Viewer 6.5 program. The origins of the gene sequences are as follows: SeNHX1 (AY131235), Salicornia europaea; AlNHX1 (GU199336.1), Aeleuropus lagopoides; AlNHX1 (AY825361.1), Aeleuropus littoralis; LfNHX1 (JF933902), Leptochloa fusca; ZjNHX1 (EU333827.2), Zoysia japonica; PaNHX1 (AB211145.1), Phragmites australis; ScNHX1 (DQ512716.1) Suaeda corniculata; SsNHX1 (EU073422), Salsola soda; SsNHX1 (AF370358.1), Suaeda salsa; SkNHX1
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For the characterization of LfNHX1 transgenic plants, osmotic stress was applied and different physiological characters were measured. Transpiration rate, stomatal conductance and net photosynthesis rate were determined using an open system LCA-4 ADC portable infrared gas analyzer (Analytical Development Co., Hoddesdon, England).
(AB531436.1), Salsola komarovii; SbNHX1 (EU448383.1), Salicornia brachiate; AhNHX1 (HM590627.1), Arachis hypogaea; ThNHX1 (AK353509.1) Thellungiella halophile; AtNHX1 (AK226586.1), Arabidopsis thaliana; HtNHX1 (EF159151.1), Helianthus tuberosus; OsNHX1 (AB021878.1), Oryza sativa, PutNHX1 (AB628206.1), Puccinellia tenuiflora; HvNHX1, (AB089197.1), Hordeum vulgare; PeNHX1 (GU295174.1), Phyllostachys edulis; TaNHX1 (AY040246.2, T. aestivum
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Fig. 2 Alignment of Cation binding domain of Na?/H? antiporter proteins. Aeleuropus lagopoides (ACZ97405.1), Aeleuropus littoralis (AAV80466.1), Leptochloa fusca (AEN03787.1), Zoysia japonica (ABY19311.2), Phragmites australis (BAD95562.1), Sorghum bicolor (ACD64982.2), Oryza sativa (AB021878.1), Arabidopsis thaliana (NP_198067.1), Puccinellia tenuiflora (BAK23261.1), Hordeum
Osmotic potential (Ws) Fully expanded youngest leaves were excised from transgenic and wild type plants. Leaf was shifted into 1.5 mL Eppendorf tube and stored at -80 °C for 2 weeks, thawed and sap was extracted by crushing with the help of micro mortar. Sap was collected and used for determination of osmotic potential in osmometer (Vapro 5520). Value obtained in mmol kg-1 was converted into—Mpa using following formula Ws ðMpaÞ ¼ 2:5 mmol kg1 =1; 000: Relative water content (RWC) Relative water contents of leaves from transgenic and wild type plants were measured according to method described by [24]. Fresh weight of equal sized discs of young and fully expanded leaves was determined after excision. Turgid weight of all discs was also measured after soaking the leaf discs in the distilled water for 22–24 h. All discs were dried carefully on tissue paper prior to measuring turgid weight. Dry weight was also measured after completely drying the discs for 72 h at 80 °C in the incubator. RWC was calculated using the following equation:
vulgare (BAC56698.1), Phyllostachys edulis (ADB54780.1), T. aestivum (AAK76738.2), Zea mays (NP_001105221.1), Lophopyrum elongatum (AAR17789.2), Atriplex dimorphostegia (AAO48271.1), Glycine max (AAY43006.1), Iris lacteal (AAU81619.1), Gossypium hirsutum (AAM54141.2)
RWC ¼ðfresh weight dry weight/turgid weight dry weightÞ 100: Leaf membrane stability index Leaf membrane stability index (MSI %) was measured according to the method described by [25] and modified by [26]. Leaf discs of uniform size and equal weight (0.2 g) were taken in 15 mL Falcon tubes containing 10 mL distilled water. Samples were kept at 40 °C in a water bath for 30 min and electrical conductivity (C1) was measured using EC meter. Another set of same size and weight was taken in test tubes and incubated at 100 °C in water bath for 15 min. Electrical conductivity (C2) of second set was also measured. MSI % was calculated by the following formula: MSI = (1 – C1/C2) 9 100. Germination analysis of transgenic and wild type plants For testing salt and Basta herbicide stress tolerance of transgenic tobacco plants, T0 seeds of transgenic and wild type plants were germinated on MS medium supplemented with 2 mg L-1 Basta and 100, 200 and 250 mM NaCl. After 10 days, resistant and wild type seedlings were transferred to MS medium supplement with 250 mM NaCl.
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Fig. 3 a Hydrophobicity plot of LfNHX1 gene product. The hydrophobicity values were calculated by the program TMpred available online at http://www.ch.embnet.org/software/TMPRED-form.html. b Proposed topological model of LfNHX1. Coiled portion indicates the 12 transmembrane domains. Third domain contain amiloride
binding site while cation binding domains is present in 5th and 6th domain. c Homology model of LfNHX1 protein based on homology with already reported vacuolar Na?/H? antiporter protein sequence. d 3-Dimensional model of LfNHX1 protein showing the 12 transmembrane domains and loops in various colors. (Color figure online)
Statistical analysis
sequence was confirmed as NHX1 sequence by NCBI sequence blast. For amplification of full length LfNHX1 cDNA, a gene specific primer and degenerate primers from 30 end were designed by alignment of NHX1 sequences from various monocot plants. A 1,300 bp fragment was amplified and confirmed by sequencing. Full length LfNHX1 gene was amplified by using gene specific primers. Full length LfNHX1 (Open reading frame) was cloned in Gateway pENTRTM TOPOÒ vector and then in expression vector by LR reaction.
Data were statistically analyzed for significance by performing analysis of variance (ANOVA) using Statistix 8.1 software. All means were compared for significance by using least significant difference test at 0.05 probability level [27]. Results Cloning of Leptochloa fusca Na?/H? antiporter gene (LfNHX1)
Bioinformatics analysis of LfNHX1 cDNA LfNHX1 (partial sequence) gene was amplified by screening L. fusca cDNA library using PCR based library screening method using M13 forward and reverse degenerate primers. A fragment of *750 bp was amplified by PCR. Amplified fragment was sequenced and putative
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LfNHX1 cDNA (Accession number: JF933902) contains an uninterrupted open reading frame of 1,623 bp coding for a polypeptide of 540 amino acids. LfNHX1gene sequence showed 95 % similarity with Aeluropus lagopoides, 88 %
Mol Biol Rep Table 1 The PROSITE patterns for LfNHX1: positions of important sites Positions
Predicted sites/patterns
Myristyl (N-myristoylation site) 13–18
GVLSST
60–65
GLCTGV
118–123
GAVGTM
121–126
GTMISF
154–159
GAIFSA
230–235 234–239
GVFPGL GLLSAY
284–289
GIVMSH
391–396
GLMRGA
CK2_phospho_site (casein kinase II phosphorylation site) 17–20
STSD
158–161
SATD
251–254
TDRE
475–478
SDLE
530–533
SPTE
ASN_glycosylation (N-glycosylation site) 51–54
NESI
294–297
NVTE
369–372
NLTK
PKC_phospho_site (protein kinase C phosphorylation site) 70–72 251–253
TTK TDR
298–300
SSR
302–304
TTK
371–373
TKK
380–382
TWR
460–462
SPK
490–492
SLR
Leucine_zipper (leucine zipper pattern) 257–278
LMMLMAYLSYMLAELSDLSGIL
ATP_GTP_A ATP/GTP-binding site motif A (P-loop) 335–342
ASDSPGKS
with Sorghum bicolor, 87 % with Zea mays, 86 with Oryza sativa, 85 % with Hordeum vulgare and 84 % with Triticum aestivum. To investigate the molecular evolution and phylogenetic relationship among Na?/H? antiporters in plants, Na?/H? antiporter gene sequences from both dicot and monocot plants were aligned and phylogenetic tree was constructed. LfNHX1 clustered with monocot halophyte plants. LfNHX1 gene belongs to the family of plant vacuolar protein which acts as Na?/H? exchanger (Fig. 1). The sequence alignment of cation binding domains showed the high similarity of LfNHX1 cation domain with cation domains from other monocot plants while there was difference of three amino acids with cation binding domains of NHX1 gene from dicot plants (Fig. 2). The
hydrophobicity plot showed that LfNHX1 has 12 hydrophobic peak regions and output from toppred suggested that LfNHX1 contains 12 strong transmembrane domains from inside to outside and outside to inside (Fig. 3a). The protein sequence 85-LFFIYLLPPI-95 in LfNHX1 protein sequence is highly conserved. This sequence was identified as amiloride binding site in mammals which inhibits eukaryotic Na?/H? antiporter. These properties confirmed that LfNHX1 is a vacuolar Na?/H? antiporter. Analysis of LfNHX1 protein sequence also showed three potential N-glycosylation sites and nine N-myristoylations sites. Furthermore, five sites of casein kinase II phosphorylation, eight protein kinase C phosphorylation sites and a leucine zipper pattern site were also predicted in LfNHX1 protein sequence (Table 1). Two dimensional secondary structure of LfNHX1 protein sequence predicted by TMRPres2D showed 12 transmembrane domains (Fig. 3b). Amiloride binding site was predicted in 3rd domain while cation binding domains was predicted in 5th and 6th domain. A structural homology model of LfNHX1 protein was obtained based on conservation scores of residues on protein structures of already reported vacuolar NHX1 protein sequences from various plant sources using ConSurf Server (Fig. 3c). Three-dimensional structure of LfNHX1 protein sequence was built using the 3D structure 4A01 chain ‘A’ as template which shares 87.5 % identities with LfNHX1 sequence. The protein data base (PDB) file of LfNHX1 protein sequence was obtained and 3D model was drawn using Chimera 1.6.2 software [22] (Fig. 3d). The sequence alignment of LfNHX1 gene with Arabidopsis, wheat and barley showed some differences in amino acid sequence. Some differences in cation binding domains were also observed (Fig. 4). LfNHX1 protein model structure was submitted to protein model database (PMDB) with PMDBID PM0078999. Construction and confirmation of transgenic tobacco plant Transgenic tobacco plants were developed by using PBLfNHX1 construct (Fig. 5a) through Agrobacterium mediated plant transformation. With the presence of bar gene in cassette, Basta was used as plant selectable marker. Putative transgenic plants were confirmed through PCR using bar gene specific primers (Fig. 5b). Southern blot analysis of transgenic and WT plants was also performed to check the gene integration. Results of Southern blot analysis showed single copy integration of the LfNHX1 gene in PB1, PB3 and PB8 transgenic lines (Fig. 5c). Beside this, reverse transcriptase PCR (RT-PCR) was also performed to check the expression of the LfNHX1 gene in the transgenic lines. RT-PCR results proved the expression of the LfNHX1 gene in all transgenic lines while there was no expression
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Fig. 4 Protein sequence alignment of LfNHX1 with other NHX from Arabidopsis thaliana (AtNHX1, AAT95387), T. aestivum (TaNHX1, AAK76738.2), Hordeum vulgare (HvNHX1, BAC56698.1). The sequences were analyzed by CLC Sequence Viewer 6.5 software.
The identical amino acid residues are indicated by black letters and different residues are indicated by white letters with black background. Small box shows amiloride binding site while large box shows cation binding domain
in wild type plant (Fig. 5d; top panel). By contrast, the house-keeping gene (NtActin) showed expression in all samples including wild type control (Fig. 5d; bottom panel). Confirmation of transgenic plant was also performed by two types of Basta leaf assays. These results showed prominent differences in transgenic and wild type plants. In case of attached Basta paint leaf assay, the circled areas where Basta herbicide was painted showed necrosis in wild type plants while transgenic plants remained unaffected (Fig. 6a). In case of detached leaf assay, leaves of transgenic plants remained green after 7 days while leaves of WT plant lost their chlorophyll (Fig. 6b).
potential as compare to wild type (Fig. 7d). It was observed that drought and heat stress decreased the membrane stability index (%) in wild type plants. Significant differences between transgenic lines and wild type plants were observed (P \ 0.05) which indicated that transgenic line possess higher membrane stability in case of drought cum heat stress (Fig. 7e).
Physiological analysis of plants for drought tolerance Transgenic plants were subjected to drought and heat stress. Plants were exposed to sunlight in net house during the summer season. Excessive heat caused rapid water loss and leaf desiccation. Holding irrigation for 7 days followed by re-watering caused great damage to the wild type plants while transgenic plants remained green and fresh even at 40 °C (Fig. 6c). Significant differences (P \ 0.05) were also observed for relative water contents, photosynthetic rate and stomatal conductance. Transgenic lines showed higher relative water contents, net photosynthetic rate and stomatal conductance as compared to wild type plants (Fig. 7a, b, c). In case of osmotic potential, transgenic plants showed significantly less negative value of osmotic
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Analysis of transgenic plants for salt and Basta herbicide tolerance Seeds of PB3 transgenic line and wild type plants were germinated on MS medium supplemented 2 mg L-1 Basta. Most of the transgenic seedlings were able to maintain their growth (Fig. 8a) while wild type seeds were unable to maintain growth and died after few days of germination (Fig. 8b). Transgenic plantlets also showed better root length at, 200 and 250 mM NaCl (Fig. 8c) while wild type plant were unable to maintain their normal growth and root length (Fig. 8d). Transgenic plant seed also showed segregation pattern showed restricted growth and behaved like wild type seed which were unable to maintain their normal growth after germination when germinated on MS0 supplemented with 2 mg L-1 Basta and 200 mM NaCl (Fig. 8d). Transgenic seedlings and wild type plant seedlings were also shifted to jars containing MS0 with 250 mM NaCl. Transgenic seedlings were able to maintain their normal shoot growth (Fig. 8e) and root length (Fig. 8g–i) while wild type seedlings showed leaf
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Fig. 5 a Diagrammatic representation of the LfNHX1 construct used for tobacco transformation. b PCR confirmation of T0 transgenic tobacco plants using bar primers. Lanes 1–6 were transgenic plants, lane 7 was wild type control and lane 8 was plasmid positive control. A 600 bp fragment of bar gene was amplified T0 transgenic lines while in wild type no amplification was detected. c Southern blot analysis of T0 transgenic plants. Lanes 1–5 were transgenic lines, lane 6 was wild-type and lane 7 was positive control. A 600 bp fragment of bar gene was used as a probe. d Reverse transcriptase PCR analysis
T0 transgenic plants showed expression of LfNHX1 gene. Gene specific primers were used to amplify 200 bp coding region of LfNHX1. NtActin gene was used as an internal control. Upper panel shows expression of LfNHX1 in transgenic tobacco. Lanes 1–6 shows 200 bps amplified PCR fragment in LfNHX1-PB transgenic lines while lane 7 represents wild type plant without amplification. Lower panel represents expression NtActin gene as internal control in transgenic as well as wild type plant (lanes 1–7) gene
chlorophyll loss and dryness (Fig. 8f) and restricted root growth (Fig. 8j) which indicated that LfNHX1 gene is working and involved in Na? extrusion mechanism.
generating proton pumps [29]. The basic salt tolerance mechanism is same in most plant species. Various Na?/H? antiporters homologs isolated from various plant species appeared to be functional for abiotic stress tolerance but cloning of Na?/H? antiporter gene homologs from halophytes would be particularly interesting because they might be functionally more efficient than those from glycophytes. Therefore research should be focused on isolation of Na?/ H? antiporters from various halophytic plant species which could survive under high saline conditions. Considering the importance of vacuolar Na?/H? antiporters in salt tolerance, it is assumed that the vacuolar Na?/H? antiporters from halophytes might have been active than those from glycophytes [30].
Discussion Drought and salinity adversely affect plant growth and limit crop yield. Plants can be classified into two groups based on salt tolerance. Glycophytes are salt sensitive while halophytes are salt tolerant plants [28, 29]. In comparison with glycophytes, halophytes have well developed mechanism for salt tolerance which may be due to the high efficiency of Na?/H? antiporter genes and electro gradient
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Fig. 6 a Leaf paint assay using 0.05 % Basta. b Detached leaf dip assay using 0.05 % Basta. c Drought stress was applied to transgenic PB3 and PB6 and wild type (WT) plants by stopping water for 7 days followed by rewatering on wilting
In this work, NHX1 homolog was isolated from a halophyte grass L. fusca by using different sets of degenerate primers. The amplified fragments were sequenced and confirmed as NHX1 gene by using NCBI blast. Bioinformatics analysis showed amino acid sequence similarity of LfNHX1 with reported vacuolar Na?/H? antiporters from various plants. LfNHX1 protein sequence showed twelve putative transmembrane domains like other plant NHX1 sequences such as SsNHX1 [31], GhNHX1 [32], ThNHX1 [33] and ZxNHX1 [34]. In the previous studies it was reported that the third transmembrane region of LfNHX1 contains the amiloride binding site (LFFIYLLPPI) which is conserved in eukaryotic Na?/H? antiporters and inhibit their activity in the presence of amiloride [31, 35–40]. This putative conserved amiloride binding domain (85LFFIYLLPPI-94) is also present in the TM3 at N-terminal of LfNHX1 protein sequence. The amino acid differences and cation binding domain indicate the differences of LfNHX1 protein sequence from AtNHX1 to TaNHX1 protein sequences. LfNHX1 gene was cloned under CaMV35S promoter using Gateway technology and tobacco transformation was done using Agrobacterium-mediated plant transformation method. Putative LfNHX1-transgenic plants were confirmed by PCR and copy number was observed using Southern hybridization whereas RT-PCR was performed for gene expression. Transgenic plants were also confirmed using Basta leaf assay. Visarada et al. [41] reported that transgenic plants can be easily identified by using leaf paint assay. Vengadesan et al. [42] reported that expression of the bar gene in transgenic plant can be confirmed by using a leaf painting test with herbicide Basta. Soliman et al. [43] also applied Basta leaf paint assay to select transgenic AtNHX1 plants containing bar gene as plant selectable marker. Results presented in this
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study are consistent with the previous reports. Some of the transgenic lines and control plant were subjected to osmotic stress by withholding water supply for 7 days during hot summer weather in the month of May when average daily day time temperature remains near 40 °C. Wild type (control) plants showed rapid leaf desiccation and growth inhibition than the transgenic plants. Brini et al. [4] and Wei et al. [44] also reported better leaf area growth and enhanced drought tolerance in case of transgenic Arabidopsis plants over-expressing TNHX1 gene from T. aestivum and AmNHX2 gene from Ammopiptanthus mongolicus, thus indicating the usability of LfNHX1under water deficit conditions. Transgenic lines showed more net photosynthetic rate and relative water contents, stomatal conductance more negative value of leaf osmotic potential and higher cell membrane stability index as compared to control plants. He et al. [45] reported higher photosynthetic rate in the transgenic cotton plants overexpressing AtNHX1 gene as compared to WT plant. Banjara et al. [46] showed enhanced photosynthetic rate and stomatal conductance in transgenic peanut overexpressing AtNHX1 gene under salt stress. Enhanced photosynthetic rate and stomatal conductance was also reported in popular plant overexpressing AtNHX1 gene under normal as well as low and high salt concentration [47]. Similarly more negative leaf water potential was observed in Arabidopsis thaliana plants overexpressing wheat Na?/H? antiporter TNHX1 gene that is in line with the findings of present study and may be due to the superior osmotic adjustment of LfNHX1 transgenic plants [4]. Transgenic plants showed higher seed germination than wild type under Basta and salt stress. Higher root and shoot length was observed in transgenic seedlings as compared to wild type. The LfNHX1-transgenic tobacco plants exhibited a better growth on 2 mg L-1 Basta while
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Fig. 7 Physiological analysis of transgenic and wild-type plants for drought tolerance. The response was measured in terms of: a relative water content RWC, b photosynthetic rate, c stomatal conductance,
d osmotic potential and e cell membrane stability index. Bars represent mean ± standard deviation
wild type plants did not maintain their survival after germination. Longer root and shoot growth was observed in transgenic plants when germinated on 100, 200 and 250 mM NaCl. This improved cellular expansion under salt stress may be due to the mediation of vacuolar K? and pH. Enhanced K?/H? exchange was also reported in case of knock out mutants of NHX1 and NHX2 genes in A. thaliana [48]. Overexpression of vacuolar AtNHX1 conferred tolerance to both salt and drought stresses in transgenic petunia plants [49]. In present study, vacuolar Na?/ H? antiporter from a halophyte grass L. fusca enhanced salt and drought tolerance in the LfNHX1-transgenic tobacco
plant. Our results suggested that LfNHX1 would be the candidate gene for engineering salt tolerance in crop plants. Many previous reports have established that H?-pyrophosphatase and Na?/H? antiporter work by complimenting each other during abiotic stress. Vacuolar proton generating pumps provide H? ions which are used by vacuolar Na?/H? antiporter for extrusion of Na? from cytoplasm into vacuole. There is need to focus on identification of H?-pyrophosphatase and Na?/H? antiporter gene homologs from halophyte plant species. Transcription factors and promoters from halophyte plants would be the best candidates to study the salt tolerance mechanisms.
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Mol Biol Rep Fig. 8 Germination test of LfNHX1 PB3 (T1) transgenic seeds (L) and wild type (R) plant seeds. a Germination of wild type (control) seeds. b Growth of transgenic seeds on MS0 supplement 2 mg L-1 Basta. c Germination of LfNHX1-PB3 transgenic seeds on MS0 with 200 mM NaCl for salt tolerance. d Germination of WT seeds on 250 mM NaCl. e Growth of transgenic seedlings on MS0 supplemented with 250 mM NaCl. f Growth of WT seedlings on 250 mM NaCl. g–i Root and shoot comparison of LfNHX1-PB3 transgenic seedlings on MS0 with 250 mM NaCl. j Growth of WT seedlings on 250 mM NaCl
Overexpressing H?-pyrophosphatase and Na?/H? antiporter gene homologs from halophyte under indigenous promoters would be helpful in generating abiotic stress tolerance in major staple food crops like wheat, maize etc. Acknowledgments The authors are also very thankful to Higher Education Commission (HEC) of Pakistan for providing PhD scholarship to Mr. Muhammad Rauf for conducting his PhD studies at NIBGE and his six months fellowship at Agricultural Biotechnology Laboratories University of Connecticut, USA.
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